Damage to inner ear hair cells is a leading cause of human deafness and balance disorders and affects >10% of the world's population. Mammals cannot regenerate their hair cells. However, birds (and other lower vertebrates) can regenerate these cells. This is a collaborative proposal from three investigators who have been pioneers in studying inner ear hair cells and applying genomic methods to their analysis. The long term goal of our research is to develop methods to regenerate and maintain inner ear hair cells. In order to achieve this goal, we first need to identify the molecular genetic pathways that will lead to sensory regeneration. Until now our insights into these processes were limited by genomic tools that were either not cost effective or were not comprehensive. With the development of high throughput Next Generation DNA sequencing methods we can now derive comprehensive digital maps of the transcriptome during the process of hair cell regeneration. We propose to use these methods to conduct a comprehensive analysis of inner ear sensory epithelia gene expression in birds and mice. We shall investigate three model systems: (1) A complete transcriptome map (mRNA and micro-RNA) of avian utricle hair cell regeneration. In addition to this we will derive a map of the majority of avian utricle-specific enhancer elements using ChIP-Seq methods and an antibody to the enhancer-associated co-factor p300. By correlating these three sets of data we expect to gain insights into which genes, which microRNAs and which long range control elements are active and important during avian hair cell regeneration. (2) The mouse utricle has a limited proliferative capacity at birth, but then loses this capacity by about two weeks after birth. We shall explore this process (again by mRNA and micro-RNA Next Gen sequencing) by comparing mouse utricles at birth (P0) and at P16 and after antibiotic killing of the hair cells at both these time points. We expect to observe differences that should provide us with insights into how these two time points differ in their proliferative capacity. (3) P0 mouse utricles treated with the gamma secretase inhibitor DAPT undergo a hyper-conversion of supporting cells to hair cells, but this capacity is lost between P12 and P16. In our third aim we will derive comprehensive mRNA and micro-RNA profiles across a DAPT treatment time course paying particular attention to the period between P12 and P16 when proliferative capacity decreases. All of the data sets derived in these studies will be validated by independent methods and all of the data will be accessible on the World Wide Web. In addition we anticipate adding a considerable degree of annotation, pathway and network analysis to the data to aid others in interpretation and utility. This project will provide three exceptionally detailed and overlapping sets of information on the development/regeneration of chicken and mouse hair cells. At the end of this project we will know all of the protein coding genes and micro-RNAs that are expressed during avian hair cell regeneration and early postnatal mouse stages under various treatments. Our groups are experienced in all of the proposed methods (and have already stockpiled many of the samples) and our goals are readily achievable within the two year time frame of this funding mechanism. PUBLIC HEALTH RELEVANCE: Damage to inner ear hair cells is a leading cause of irreversible human hearing loss and balance disorders, which affect >10% of the world's population. Mammals cannot regenerate their hair cells, however, birds (and other lower vertebrates) can regenerate these cells and recover hearing and balance functions. The long-term goal of our research is to develop methods to regenerate and maintain inner ear hair cells by studying the regenerative process in birds and comparing it to mammalian hair cell growth.